1. Field of Invention:
This invention is directed to a method and a diagnostic kit. More specifically, the invention described hereinafter relates to a method for the detection of serum antibodies to microbial antigens and/or the direct detection and quantitiation of these microbial antigens by radial partition immunoassay and a test kit for the performance of such assay.
2. Description of the Prior Art:
(a) A number of the advances in the field of diagnosis of infectious disease attributable to microganisms, have been made as a direct result of automation of the microbial identification process. These advances have not only reduced the turn-around time for performance of such analysis, but have increased the accuracy of results which can be attained. The major difficulties encountered in the improvement of such assays is the need to reduce the processing interval for performance of the assay. Classically, microbe detection has involved considerable delay and expense traditionally associated with manual techniques, incubation steps, irreproducibility and unreliability of results. It has been both the hope and objective of the developers of semi- and essentially fully automated systems, that these deficiencies in the classical methods can be eliminated or minimized through the adaptation of automation to this art.
Semi-automated systems are currently commercially available for performance of the above type assay. Typically these systems employ some kind of support media (i.e., wells, cups or tubes) into which is placed an aliquot of patient sample. Added to this at different stages are signal generating reagent(s). After several sets of washings and one or more periods of incubation, a reaction of this analyte in the sample with the signal generating reagent(s) occurs. The activity of the signal generating reagent(s) is related to the amount of the analyte present in the sample. The signal measured may be a radiolabeled, a chromogenic or a fluorogenic compound which can be detected by instrumentation; or, observed visually through comparison of color change with a set of reference colors specific for the given substance being tested.
Since the rapid detection and accurate identification of the microbial species is the predominant concern of the clinical microbioloby laboratory, most of the automated systems which have been introduced are designed to reduce the amount of required manipulative steps, and also increase the efficiency of the assay process. Notwithstanding such advances, significant limitations in the state of the art still exist. Representation of one such commercially available system is MINITEK.RTM. microorganism differentiation system (available from Becton, Dickinson and Company). This MINITEK system uses a plate containing ten (10) wells, into which are added one or more cartridge-dispensed paper discs impregnated with a substrate. A microbe colony, isolated from an incubated culture, is also added to the well. The contents of the well is then covered with a layer of mineral oil to prevent contamination. The contents are thereafter incubated for 18-24 hours. Then, as applicable, reagents are added and allowed to react with the contents of the well; the results of such interaction ultimately being determined by comparison of color changes in the liquid in the well with the appropriate reference comparator card. One of the more significant limitations of this MINITEK system as with most others which are currently being used, is the delay encountered before results can be obtained. The system requires two extended incubation periods, one to grow the microbe, and a second to allow the sample and substrate to react. The MINITEK system also is highly labor intensive, requiring several manual manipulative steps. Furthermore, the system must be used with pure cultures, it being incompatible with specimens taken directly from the patient since the presence of endogenous interactive substances in the patient sample can interfere with the accuracy of the assay. The assay results obtained from the tests performed on this system are limited in precision to a positive/negative determination or to a range of color-compared variation.
(b) Antimicrobial Sensitivity Assay techniques have been developed as a means for rapidly determining the most efficacious drug to be used in the treatment of a particular infection without the need for obtaining precise identification of the bacteria causing the infection. This method comtemplates observing which of a set of drugs best inhibits the growth in vitro of bacteria contained in a patient sample. the primary objective of this screening technique is to provide the physician with an accurate, fast and reliable basis for his choice of therapeutic agent.
Traditional anti-microbial sensitivity assay techniques are routinely accomplished by obtaining a patient sample containing the infectious agent and adding the sample to a series of test tubes containing a growth media. In all but one of the test tubes (a control), are added different antibiotics (one per tube) which are potentially useful for treatment of the individual affected with the infectious agent. Alternatively, sensitivity testing may be performed by using a petri dish containing growth media onto which the bacterium being tested is streaked. Following the placement of a series of impregnated antibiotic-containing "disc's" onto the surface of this medium, the drug of choice is determined by measuring the amount of growth after a predetermined incubation period. As with the test tube method, the region about the antibiotic showing the greatest degree of clearing (i.e., demonstrating the least amount of bacterial growth) is indicative of which drug has best inhibited the growth of the microbe. This drug is thus the drug of choice in treatment of the microbial infection. The method of measuring the degree of growth of the infectious agent in a patient sample in the presence of different drugs is the subject of this invention.
(c,d) A technique currently used for microbial antigen detection and identification is that of ELISA, or enzyme-linked immunosorbent assay. In a classical heterogeneous assay (such as described in U.S. Pat. No. 3,654,090), microbial identification is performed in a series of microtiter wells coated with antibody. This type of assay generally involves at least two incubation periods and several wash steps. Here, too, there are numerous limitations associated with the system. As with the microoganism identification systems discussed above, two incubation periods are required. Many of the other limitations and deficiencies associated with the microorganism identification systems discussed hereinabove are also present in microbial identification by an ELISA technique. Moreover, the typical microtiter plate consists of ninety-six (96) wells. If the number of assays to be performed is less than the total number of wells, the cost per test is increased proportionally.
The problems and shortcomings associated with the techniques described hereinabove are encountered to a similar extent in microbial identification assays, the determination/quantitation of viral surface antigen, and the corresponding detection of serum antibodies against these microbial antigens.
As is evident from the above discussion, there is a continuing need for improvement in the currently available techniques for surface antigen detection in the following respects: reduction in the number of manipulative steps which are performed manually by the tehnician; reduction in the time required for the performance of such assays; enhancement in the accuracy of the reliability of the test results; and, reduction in the cost per test charge to the patient.